RNAi-MEDIATED INHIBITION OF TUMOR NECROSIS FACTOR ALPHA-RELATED CONDITIONS

ABSTRACT

RNA interference is provided for inhibition of tumor necrosis factor α (TNFα) by silencing TNFα cell surface receptor TNF receptor-1 (TNFR1) mRNA expression, or by silencing TNFα converting enzyme (TACE/ADAM17) mRNA expression. Silencing such TNFα targets, in particular, is useful for treating patients having a TNFα-related condition or at risk of developing a TNFα-related condition such as the ocular conditions dry eye, allergic conjunctivitis, or ocular inflammation, or such as dermatitis, rhinitis, or asthma, for example.

RELATED APPLICATION

The present application is a divisional application of U.S. patentapplication Ser. No. 13/035,147 filed Feb. 25, 2011, which claimsbenefit to U.S. patent application Ser. No. 12/700,188 filed Feb. 4,2010, which claims benefit to U.S. patent application Ser. No.11/750,262 filed May 17, 2007 (now U.S. Pat. No. 7,732,421), whichclaims benefit to U.S. Provisional Patent Application Ser. No.60/801,788 filed on May, 19, 2006, titled RNAi-MEDIATED INHIBITION OFTUMOR NECROSIS FACTOR α-RELATED CONDITIONS, the text of which isspecifically incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the field of interfering RNAcompositions for silencing tumor necrosis factor α (TNFα) by silencingthe TNFα cell surface receptor TNF receptor-1 (TNFR1) mRNA, or the TNFαconverting enzyme (TACE/ADAM17) mRNA. Silencing such TNFα targets isuseful for treatment of patients having a TNFα-related condition or atrisk of developing such a condition.

BACKGROUND OF THE INVENTION

Inflammation is generally treated with a standard anti-inflammatoryregimen that includes steroids and/or non-steroidal anti-inflammatorydrugs (NSAIDS). Allergic conjunctivitis, ocular inflammation,dermatitis, rhinitis, and asthma have historically been treated with aregimen of oral, intranasal or topical antihistamines in addition to ororal or intranasal steroids. Systemic treatment typically requireshigher concentrations of the drug compound to be administered to affordan effective concentration to reach the necessary treatment site.Antihistamine compounds are known to have central nervous systemactivity; drowsiness and drying of mucus membranes are a commonside-effect of antihistamine use. Steroids and NSAIDS have potentialside effects including intraocular pressure increase, cataract, glaucomaor corneal melting.

Dry eye, also known as conjunctivitis sicca or keratoconjunctivitissicca, is a common ophthalmological disorder involving breakdown of thepre-ocular tear film, resulting in dehydration of the exposed outersurface of the eye. To date, dry eye has been treated with topicaladministration of artificial tear solutions. Some of these solutionscontain mucomimetic substances to temporarily replace or replenish themucin layer in mucin deficient patients. Use of methylprednisolone hasbeen proposed in a short-term “pulse” treatment to treat exacerbationsof dry eye. The proposed “pulse” therapy is required to avoidcomplications associated with traditional steroid therapy forinflammatory conditions such as increased intraocular pressure andcataract formation.

The cytokine TNFα is a target for anti-inflammatory therapy of dry eyeand uveitis. In a rabbit model of lacrimal gland inflammation-induceddry eye, inhibition of corneal staining and restoration of tear breakuptime has been achieved by specific modulation of ocular surface TNFαlevels. Dry eye therapy resulted by inhibiting TNFα synthesis (RDP58) orby specifically neutralizing TNFα using a monoclonal antibody(REMICADE®) or a soluble receptor (ENBREL®). Each of these TNFα directedtreatments resulted in levels of efficacy obtained with topical ocularanti-inflammatory steroids.

U.S. Patent Publication 2005/0227935, published Oct. 13, 2005, toMcSwiggen et al. relates to RNA interference mediated inhibition of TNFand TNF receptor gene expression. However, said publication teaches noneof the particular target sequences for RNA interference as providedherein.

Embodiments of the present invention address the need in the art forfurther agents and treatment methods for dry eye and inflammation andprovide alternative therapies therefor.

SUMMARY OF THE INVENTION

Embodiments of the present invention provide highly potent andefficacious treatment, prevention or intervention of a TNFα-relatedcondition without side effects associated with steroids or NSAIDS. Inone aspect, methods of the invention include treating a subject having aTNFα-related condition or at risk of developing a TNFα-related conditionby administering interfering RNAs that silence expression of TACE mRNAor TNFR1 mRNA, thus interfering with proteolytic processing of theprecursor to TNFα, or interfering with binding of TNFα to its cellsurface receptor, respectively, thereby attenuating activity of TNFα andpreventing a cascade of events related to apoptosis and inflammation.

A TNFα-related condition includes conditions such as dry eye andTNFα-related inflammatory conditions. A TNFα-related inflammatorycondition includes conditions such as ocular inflammation, allergicconjunctivitis, dermatitis, rhinitis, and asthma, for example, andincludes those cellular changes resulting from the activity of TNFα thatleads directly or indirectly to the TNFα-related inflammatory condition.A TNFα-related condition particularly includes TNFα-related ocularconditions such as dry eye, allergic conjunctivitis, and ocularinflammation. The interfering RNA provided herein provides for silencingthe TNFα targets TACE mRNA or TNFR1 mRNA while avoiding undesirable sideeffects due to nonspecific agents.

A method of attenuating expression of TACE mRNA of the subject is anembodiment of the invention. The method comprises administering to thesubject a composition comprising an effective amount of interfering RNAhaving a length of 19 to 49 nucleotides and a pharmaceuticallyacceptable carrier, the interfering RNA comprising a region of at least13 contiguous nucleotides having at least 90% sequence complementarityto, or at least 90% sequence identity with, the penultimate 13nucleotides of the 3′ end of an mRNA corresponding to any one of SEQ IDNO:3, SEQ ID NO:14-SEQ ID NO:58, and SEQ ID NO:155-SEQ ID NO:201. Theexpression of TACE mRNA is attenuated thereby.

A method of treating a TNFα-related condition in a subject in needthereof is an embodiment of the invention. The method comprisesadministering to the subject a composition comprising an effectiveamount of interfering RNA having a length of 19 to 49 nucleotides, and apharmaceutically acceptable carrier, wherein the interfering RNAcomprises a region of at least 13 contiguous nucleotides having at least90% sequence complementarity to, or at least 90% sequence identity with,the penultimate 13 nucleotides of the 3′ end of an mRNA corresponding toany one of SEQ ID NO:3, SEQ ID NO:14-SEQ ID NO:58, and SEQ ID NO:155-SEQID NO:201. The TNFα-related condition is treated thereby.

In yet another embodiment of the invention, a method of attenuatingactivity of TNFα of a subject by attenuating expression of TACE mRNA orTNFR1 mRNA of the subject comprises administering to the subject acomposition comprising an effective amount of interfering RNA having alength of 19 to 49 nucleotides and a pharmaceutically acceptable carrierand the interfering RNA comprises a sense nucleotide strand, anantisense nucleotide strand, and a region of at least near-perfectcontiguous complementarity of at least 19 nucleotides where theantisense strand hybridizes under physiological conditions to a portionof mRNA corresponding to SEQ ID NO:1 comprising nucleotide 297, 333,334, 335, 434, 470, 493, 547, 570, 573, 618, 649, 689, 755, 842, 844,846, 860, 878, 894, 900, 909, 910, 913, 942, 970, 984, 1002, 1010, 1053,1064, 1137, 1162, 1215, 1330, 1334, 1340, 1386, 1393, 1428, 1505, 1508,1541, 1553, 1557, 1591, 1592, 1593, 1597, 1604, 1605, 1626, 1632, 1658,1661, 1691, 1794, 1856, 1945, 1946, 1947, 1958, 2022, 2094, 2100, 2121,2263, 2277, 2347, 2349, 2549, 2578, 2595, 2606, 2608, 2629, 2639, 2764,2766, 2767, 2769, 3027, 3028, 3261, 3264, 3284, 3313, 3317, 3332, or3337 or where the antisense strand hybridizes under physiologicalconditions to a portion of mRNA corresponding to SEQ ID NO:2 beginningat nucleotide 124, 328, 387, 391, 393, 395, 406, 421, 423, 444, 447,455, 459, 460, 467, 469, 470, 471, 475, 479, 513, 517, 531, 543, 556,576, 587, 588, 589, 595, 601, 602, 611, 612, 651, 664, 667, 668, 669,677, 678, 785, 786, 788, 791, 792, 804, 813, 824, 838, 843, 877, 884,929, 959, 960, 961, 963, 964, 965, 970, 973, 974, 1000, 1002, 1013,1026, 1053, 1056, 1057, 1058, 1161, 1315, 1318, 1324, 1357, 1360, 1383,1393, 1420, 1471, 1573, 1671, 2044, 2045, 2046, 2047, 2048, 2089, 2090,2091, 2092, or 2098. The expression of TACE mRNA is attenuated in thoseembodiments where the antisense stand hybridizes to a portion of mRNAcorresponding to SEQ ID NO:1 as cited above. The expression of TNFR1mRNA is attenuated in those embodiments where the antisense standhybridizes to a portion of mRNA corresponding to SEQ ID NO:2 as citedabove.

A method of treating a TNFα-related condition in a subject in needthereof is an embodiment of the invention, the method comprisingadministering to the subject a composition comprising an effectiveamount of interfering RNA having a length of 19 to 49 nucleotides, and apharmaceutically acceptable carrier, the interfering RNA comprising asense nucleotide strand, an antisense nucleotide strand, and a region ofat least near-perfect contiguous complementarity of at least 19nucleotides; wherein the antisense strand hybridizes under physiologicalconditions to a portion of mRNA corresponding to SEQ ID NO:1 comprisingnucleotide 297, 333, 334, 335, 434, 470, 493, 547, 570, 573, 618, 649,689, 755, 842, 844, 846, 860, 878, 894, 900, 909, 910, 913, 942, 970,984, 1002, 1010, 1053, 1064, 1137, 1162, 1215, 1330, 1334, 1340, 1386,1393, 1428, 1505, 1508, 1541, 1553, 1557, 1591, 1592, 1593, 1597, 1604,1605, 1626, 1632, 1658, 1661, 1691, 1794, 1856, 1945, 1946, 1947, 1958,2022, 2094, 2100, 2121, 2263, 2277, 2347, 2349, 2549, 2578, 2595, 2606,2608, 2629, 2639, 2764, 2766, 2767, 2769, 3027, 3028, 3261, 3264, 3284,3313, 3317, 3332, or 3337. The TNFα-related condition is treatedthereby.

A method of treating a TNFα-related ocular condition in a subject inneed thereof is an embodiment of the invention, the method comprisingadministering to the subject a composition comprising an effectiveamount of interfering RNA having a length of 19 to 49 nucleotides, and apharmaceutically acceptable carrier, the interfering RNA comprising asense nucleotide strand, an antisense nucleotide strand, and a region ofat least near-perfect contiguous complementarity of at least 19nucleotides; wherein the antisense strand hybridizes under physiologicalconditions to a portion of mRNA corresponding to SEQ ID NO:2 comprisingnucleotide 124, 328, 387, 391, 393, 395, 406, 421, 423, 444, 447, 455,459, 460, 467, 469, 470, 471, 475, 479, 513, 517, 531, 543, 556, 576,587, 588, 589, 595, 601, 602, 611, 612, 651, 664, 667, 668, 669, 677,678, 785, 786, 788, 791, 792, 804, 813, 824, 838, 843, 877, 884, 929,959, 960, 961, 963, 964, 965, 970, 973, 974, 1000, 1002, 1013, 1026,1053, 1056, 1057, 1058, 1161, 1315, 1318, 1324, 1357, 1360, 1383, 1393,1420, 1471, 1573, 1671, 2044, 2045, 2046, 2047, 2048, 2089, 2090, 2091,2092, or 2098. The TNFα-related condition is treated thereby.

A second interfering RNA having a length of 19 to 49 nucleotides couldalso be administered to the subject in a further embodiment; the secondinterfering RNA comprising a sense nucleotide strand, an antisensenucleotide strand, and a region of at least near-perfect complementarityof at least 19 nucleotides wherein the antisense strand of the secondinterfering RNA hybridizes under physiological conditions to a portionof mRNA corresponding to SEQ ID NO:1 comprising a nucleotide as citedabove, or where the antisense strand hybridizes under physiologicalconditions to a portion of mRNA corresponding to SEQ ID NO:2 beginningat nucleotide 124, 328, 387, 391, 393, 395, 406, 421, 423, 444, 447,455, 459, 460, 467, 469, 470, 471, 475, 479, 513, 517, 531, 543, 556,576, 587, 588, 589, 595, 601, 602, 611, 612, 651, 664, 667, 668, 669,677, 678, 785, 786, 788, 791, 792, 804, 813, 824, 838, 843, 877, 884,929, 959, 960, 961, 963, 964, 965, 970, 973, 974, 1000, 1002, 1013,1026, 1053, 1056, 1057, 1058, 1161, 1315, 1318, 1324, 1357, 1360, 1383,1393, 1420, 1471, 1573, 1671, 2044, 2045, 2046, 2047, 2048, 2089, 2090,2091, 2092, or 2098.

When a first interfering RNA targets SEQ ID NO:1, the second interferingRNA may target either SEQ ID NO:1 or SEQ ID NO:2, and conversely, when afirst interfering RNA targets SEQ ID NO:2, the second interfering RNAmay target either SEQ ID NO:1 or SEQ ID NO:2. In further embodiments, athird, fourth, or more interfering RNAs may be administered.

A further embodiment of the invention is a method of treating aTNFα-related condition in a subject in need thereof, where the methodcomprises administering to the subject a composition comprising a doublestranded siRNA molecule that down regulates expression of a TACE genevia RNA interference, wherein each strand of the siRNA molecule isindependently about 19 to about 27 nucleotides in length; and one strandof the siRNA molecule comprises a nucleotide sequence having substantialcomplementarity to an mRNA corresponding to the TACE gene, so that thesiRNA molecule directs cleavage of the mRNA via RNA interference.

A further embodiment of the invention is a method of treating aTNFα-related ocular condition in a subject in need thereof, where themethod comprises administering to the subject a composition comprising adouble stranded siRNA molecule that down regulates expression of a TNFR1gene via RNA interference, wherein each strand of the siRNA molecule isindependently about 19 to about 27 nucleotides in length; and one strandof the siRNA molecule comprises a nucleotide sequence having substantialcomplementarity to an mRNA corresponding to the TNFR1 gene so that thesiRNA molecule directs cleavage of the mRNA via RNA interference.

A method of attenuating expression of TACE mRNA of the subject,comprising administering to the subject a composition comprising aneffective amount of a single-stranded interfering RNA and apharmaceutically acceptable carrier is a further embodiment. Thesingle-stranded interfering RNA has a length of 19 to 49 nucleotides andhybridizes under physiological conditions to a portion of mRNAcorresponding to SEQ ID NO:1 comprising nucleotide 297, 333, 334, 335,434, 470, 493, 547, 570, 573, 618, 649, 689, 755, 842, 844, 846, 860,878, 894, 900, 909, 910, 913, 942, 970, 984, 1002, 1010, 1053, 1064,1137, 1162, 1215, 1330, 1334, 1340, 1386, 1393, 1428, 1505, 1508, 1541,1553, 1557, 1591, 1592, 1593, 1597, 1604, 1605, 1626, 1632, 1658, 1661,1691, 1794, 1856, 1945, 1946, 1947, 1958, 2022, 2094, 2100, 2121, 2263,2277, 2347, 2349, 2549, 2578, 2595, 2606, 2608, 2629, 2639, 2764, 2766,2767, 2769, 3027, 3028, 3261, 3264, 3284, 3313, 3317, 3332, or 3337, andthe interfering RNA has a region of at least near-perfect contiguouscomplementarity with the hybridizing portion of mRNA corresponding toSEQ ID NO: 1. The expression of TACE mRNA is thereby attenuated.

The invention includes as a further embodiment a composition comprisingan interfering RNA having a length of 19 to 49 nucleotides, andcomprising a nucleotide sequence corresponding to any one of SEQ IDNO:3, SEQ ID NO:14-SEQ ID NO:58, and SEQ ID NO:155-SEQ ID NO:201, or acomplement thereof; and a pharmaceutically acceptable carrier.

The invention includes as a further embodiment a composition comprisingan interfering RNA consisting essentially of a nucleotide sequencecorresponding to any one of SEQ ID NO:59-SEQ ID NO:69, SEQ ID NO:71-SEQID NO:92, and SEQ ID NO:94-SEQ ID NO:154, or a complement thereof; and apharmaceutically acceptable carrier.

Use of any of the embodiments as described herein in the preparation ofa medicament for attenuating expression of TACE mRNA or of TNFR1 mRNA asa method of attenuating activity of TNFα and thereby treating aTNFα-related condition as set forth herein is also an embodiment of thepresent invention.

BRIEF DESCRIPTION OF THE FIGURES

In order that the manner in which the above recited and other advantagesand objects of the invention are obtained, a more particular descriptionof the invention briefly described above will be rendered by referenceto specific embodiments thereof, which are illustrated, in the appendeddrawings. Understanding that these drawings depict only typicalembodiments of the invention and are therefore not to be consideredlimiting of its scope, the invention will be described with additionalspecificity and detail through the use of the accompanying drawings inwhich:

FIG. 1 provides a TNFR1 western blot of GTM-3 cells transfected withTNFR1 siRNAs #1, #2, #3, and #4, and a RISC-free control siRNA, each at10 nM, 1 nM, and 0.1 nM; a non-targeting control siRNA (NTC2) at 10 nM;and a buffer control (-siRNA). The arrows indicate the positions of the55-kDa TNFR1 and 42-kDa actin bands.

DETAILED DESCRIPTION OF THE INVENTION

The references cited herein, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated by reference.

Those of skill in the art, in light of the present disclosure, willappreciate that obvious modifications of the embodiments disclosedherein can be made without departing from the spirit and scope of theinvention. All of the embodiments disclosed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. The full scope of the invention is set out in the disclosureand equivalent embodiments thereof. The specification should not beconstrued to unduly narrow the full scope of protection to which thepresent invention is entitled.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention may be embodied in practice.

The following definitions and explanations are meant and intended to becontrolling in any future construction unless clearly and unambiguouslymodified in the following examples or when application of the meaningrenders any construction meaningless or essentially meaningless. Incases where the construction of the term would render it meaningless oressentially meaningless, the definition should be taken from Webster'sDictionary, 3^(rd) Edition.

As used herein, all percentages are percentages by weight, unless statedotherwise.

As used herein and unless otherwise indicated, the terms “a” and “an”are taken to mean “one”, “at least one” or “one or more”.

The term “dry eye,” also known as conjunctivitis sicca orkeratoconjunctivitis sicca, is a common ophthalmological disorderinvolving breakdown of the pre-ocular tear film, resulting indehydration of the exposed outer surface of the eye.

The term “ocular inflammation,” as used herein, includes iritis,uveitis, episcleritis, scleritis, keratitis, endophthalmitis,blepharitis, and iatrogenic inflammatory conditions, for example.

The term “allergic conjunctivitis,” as used herein, refers toinflammation of the conjunctiva which is the delicate membrane thatlines the eyelids and covers the exposed surface of the sclera. The term“allergic conjunctivitis” includes, for example, atopickeratoconjunctivitis, giant papillary conjunctivitis, hay feverconjunctivitis, perennial allergic conjunctivitis, and vernalkeratoconjunctivitis.

The term “dermatitis,” as used herein, refers to inflammation of theskin and includes, for example, allergic contact dermatitis, urticaria,asteatotic dermatitis (dry skin on the lower legs), atopic dermatitis,contact dermatitis including irritant contact dermatitis andurushiol-induced contact dermatitis, eczema, gravitational dermatitis,nummular dermatitis, otitis externa, perioral dermatitis, andseborrhoeic dermatitis.

The term “rhinitis,” as used herein, refers to inflammation of themucous membranes of the nose and includes, for example, allergicrhinitis, atopic rhinitis, irritant rhinitis, eosinophilic non-allergicrhinitis, rhinitis medicamentosa, and neutrophilic rhinosinusitis.

The term “asthma,” as used herein, refers to inflammation of the airpassages resulting in narrowing of the airways that transport air fromthe nose and mouth to the lungs and includes, for example, allergicasthma, atopic asthma, atopic bronchial IgE-mediated asthma, bronchialasthma, bronchiolytis, emphysematous asthma, essential asthma,exercise-induced asthma, extrinsic asthma caused by environmentalfactors, incipient asthma, intrinsic asthma caused by pathophysiologicdisturbances, non-allergic asthma, non-atopic asthma, and wheezy infantsyndrome.

The phrase “a region of at least 13 contiguous nucleotides having atleast 90% sequence complementarity to, or at least 90% sequence identitywith, the penultimate 13 nucleotides of the 3′ end of an mRNAcorresponding to any one of (a sequence identifier)” allows a onenucleotide substitution. Two nucleotide substitutions (i.e., 11/13=85%identity/complementarity) are not included in such a phrase.

The term “percent identity” describes the percentage of contiguousnucleotides in a first nucleic acid molecule that is the same as in aset of contiguous nucleotides of the same length in a second nucleicacid molecule. The term “percent complementarity” describes thepercentage of contiguous nucleotides in a first nucleic acid moleculethat can base pair in the Watson-Crick sense with a set of contiguousnucleotides in a second nucleic acid molecule.

As used herein, the term “hybridization” means and refers to a processin which single-stranded nucleic acids with complementary ornear-complementary base sequences interact to form hydrogen-bondedcomplexes called hybrids. Hybridization reactions are sensitive andselective. In vitro, the specificity of hybridization (i.e., stringency)is controlled by the concentrations of salt or formamide inprehybridization and hybridization solutions, for example, and by thehybridization temperature; such procedures are well known in the art. Inparticular, stringency is increased by reducing the concentration ofsalt, increasing the concentration of formamide, or raising thehybridization temperature.

For example, high stringency conditions could occur at about 50%formamide at 37° C. to 42° C. Reduced stringency conditions could occurat about 35% to 25% formamide at 30° C. to 35° C. Examples of stringencyconditions for hybridization are provided in Sambrook, J., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. Further examples of stringenthybridization conditions include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing, orhybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamidefollowed by washing at 70° C. in 0.3×SSC, or hybridization at 70° C. in4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at 67° C. in1×SSC. The temperature for hybridization is about 5-10° C. less than themelting temperature (T_(m)) of the hybrid where T_(m) is determined forhybrids between 19 and 49 base pairs in length using the followingcalculation: T_(m)° C.=81.5+16.6(log₁₀[Na+])+0.41 (% G+C)−(600/N) whereN is the number of bases in the hybrid, and [Na+] is the concentrationof sodium ions in the hybridization buffer.

Nucleic acid sequences cited herein are written in a 5′ to 3′ directionunless indicated otherwise. The term “nucleic acid,” as used herein,refers to either DNA or RNA or a modified form thereof comprising thepurine or pyrimidine bases present in DNA (adenine “A,” cytosine “C,”guanine “G,” thymine “T”) or in RNA (adenine “A,” cytosine “C,” guanine“G,” uracil “U”). Interfering RNAs provided herein may comprise “T”bases, particularly at 3′ ends, even though “T” bases do not naturallyoccur in RNA. “Nucleic acid” includes the terms “oligonucleotide” and“polynucleotide” and can refer to a single-stranded molecule or adouble-stranded molecule. A double-stranded molecule is formed byWatson-Crick base pairing between A and T bases, C and G bases, andbetween A and U bases. The strands of a double-stranded molecule mayhave partial, substantial or full complementarity to each other and willform a duplex hybrid, the strength of bonding of which is dependent uponthe nature and degree of complementarity of the sequence of bases.

An mRNA sequence is readily deduced from the sequence of thecorresponding DNA sequence. For example, SEQ ID NO:1 provides the sensestrand sequence of DNA corresponding to the mRNA for TACE. The mRNAsequence is identical to the DNA sense strand sequence with the “T”bases replaced with “U” bases. Therefore, the mRNA sequence of TACE isknown from SEQ ID NO:1 and the mRNA of TNFR1 is known from SEQ ID NO:2.

RNA interference (RNAi) is a process by which double-stranded RNA(dsRNA) is used to silence gene expression. While not wanting to bebound by theory, RNAi begins with the cleavage of longer dsRNAs intosmall interfering RNAs (siRNAs) by an RNaseIII-like enzyme, dicer.SiRNAs are dsRNAs that are usually about 19 to 28 nucleotides, or 20 to25 nucleotides, or 21 to 22 nucleotides in length and often contain2-nucleotide 3′ overhangs, and 5′ phosphate and 3′ hydroxyl termini. Onestrand of the siRNA is incorporated into a ribonucleoprotein complexknown as the RNA-induced silencing complex (RISC). RISC uses this siRNAstrand to identify mRNA molecules that are at least partiallycomplementary to the incorporated siRNA strand, and then cleaves thesetarget mRNAs or inhibits their translation. Therefore, the siRNA strandthat is incorporated into RISC is known as the guide strand or theantisense strand. The other siRNA strand, known as the passenger strandor the sense strand, is eliminated from the siRNA and is at leastpartially homologous to the target mRNA. Those of skill in the art willrecognize that, in principle, either strand of an siRNA can beincorporated into RISC and function as a guide strand. However, siRNAdesign (e.g., decreased siRNA duplex stability at the 5′ end of theantisense strand) can favor incorporation of the antisense strand intoRISC.

RISC-mediated cleavage of mRNAs having a sequence at least partiallycomplementary to the guide strand leads to a decrease in the steadystate level of that mRNA and of the corresponding protein encoded bythis mRNA. Alternatively, RISC can also decrease expression of thecorresponding protein via translational repression without cleavage ofthe target mRNA. Other RNA molecules and RNA-like molecules can alsointeract with RISC and silence gene expression. Examples of other RNAmolecules that can interact with RISC include short hairpin RNAs(shRNAs), single-stranded siRNAs, microRNAs (miRNAs), anddicer-substrate 27-mer duplexes. The term “siRNA” as used herein refersto a double-stranded interfering RNA unless otherwise noted. Examples ofRNA-like molecules that can interact with RISC include RNA moleculescontaining one or more chemically modified nucleotides, one or moredeoxyribonucleotides, and/or one or more non-phosphodiester linkages.For purposes of the present discussion, all RNA or RNA-like moleculesthat can interact with RISC and participate in RISC-mediated changes ingene expression will be referred to as “interfering RNAs.” SiRNAs,shRNAs, miRNAs, and dicer-substrate 27-mer duplexes are, therefore,subsets of “interfering RNAs.”

Interfering RNA of embodiments of the invention appear to act in acatalytic manner for cleavage of target mRNA, i.e., interfering RNA isable to effect inhibition of target mRNA in substoichiometric amounts.As compared to antisense therapies, significantly less interfering RNAis required to provide a therapeutic effect under such cleavageconditions.

The present invention relates to the use of interfering RNA to inhibitthe expression of TNFα cell surface receptor TNF receptor-1 (TNFR1), orthe TNFα converting enzyme (TACE/ADAM17, designated herein “TACE”) whichinhibition effects reduction of tumor necrosis factor α (TNFα) activity.Binding of TNFα to its cell surface receptor, TNF receptor-1 (TNFR1),activates a signaling cascade which affects a variety of cellularresponses including apoptosis and inflammation. TNFα itself is initiallyexpressed as an inactive, membrane-bound precursor. Release of theactive form of TNFα from the cell surface requires proteolyticprocessing of the precursor by TNFα converting enzyme (TACE/ADAM17), amember of the ‘A Disintegrin And Metalloprotease’ (ADAM) family.

According to the present invention, inhibiting the expression of TNFR1mRNA, TACE mRNA, or both TNFR1 and TACE mRNAs effectively reduces theaction of TNFα. Further, interfering RNAs as set forth herein providedexogenously or expressed endogenously are particularly effective atsilencing TNFR1 mRNA or TACE mRNA.

Tumor Necrosis Factor α Converting Enzyme mRNA (TACE/ADAM17):

The GenBank database provides the DNA sequence for TACE as accession no.NM 003183, provided in the “Sequence Listing” as SEQ ID NO:1. SEQ IDNO:1 provides the sense strand sequence of DNA that corresponds to themRNA encoding TACE (with the exception of “T” bases for “U” bases). Thecoding sequence for TACE is from nucleotides 184-2658.

Equivalents of the above cited TACE mRNA sequence are alternative spliceforms, allelic forms, isozymes, or a cognate thereof. A cognate is atumor necrosis factor α converting enzyme mRNA from another mammalianspecies that is homologous to SEQ ID NO:1 (i.e., an ortholog).

Tumor Necrosis Factor Receptor-1 mRNA (TNFR1):

The GenBank database provides the DNA sequence for TNFR1 as accessionno. NM 001065, provided in the “Sequence Listing” as SEQ ID NO:2. SEQ IDNO:2 provides the sense strand sequence of DNA that corresponds to themRNA encoding TNFR1 (with the exception of “T” bases for “U” bases). Thecoding sequence for TNFR1 is from nucleotides 282-1649.

Equivalents of the above cited TNFR1 mRNA sequence are alternativesplice forms, allelic forms, isozymes, or a cognate thereof. A cognateis a tumor necrosis factor receptor-1 mRNA from another mammalianspecies that is homologous to SEQ ID NO:2 (i.e., an ortholog).

Attenuating Expression of an mRNA:

The phrase, “attenuating expression of an mRNA,” as used herein, meansadministering or expressing an amount of interfering RNA (e.g., ansiRNA) to reduce translation of the target mRNA into protein, eitherthrough mRNA cleavage or through direct inhibition of translation. Thereduction in expression of the target mRNA or the corresponding proteinis commonly referred to as “knock-down” and is reported relative tolevels present following administration or expression of a non-targetingcontrol RNA (e.g., a non-targeting control siRNA). Knock-down ofexpression of an amount including and between 50% and 100% iscontemplated by embodiments herein. However, it is not necessary thatsuch knock-down levels be achieved for purposes of the presentinvention. In one embodiment, a single interfering RNA targeting TACEmRNA or TNFR1 mRNA is administered. In other embodiments, two or moreinterfering RNAs targeting TACE mRNA or TNFR1 mRNA are administered. Infurther embodiments, interfering RNAs targeting each of TACE mRNA andTNFR1 mRNA are administered in combination or in a time interval so asto have overlapping effects.

Knock-down is commonly assessed by measuring the mRNA levels usingquantitative polymerase chain reaction (qPCR) amplification or bymeasuring protein levels by western blot or enzyme-linked immunosorbentassay (ELISA). Analyzing the protein level provides an assessment ofboth mRNA cleavage as well as translation inhibition. Further techniquesfor measuring knock-down include RNA solution hybridization, nucleaseprotection, northern hybridization, gene expression monitoring with amicroarray, antibody binding, radioimmunoassay, and fluorescenceactivated cell analysis.

Inhibition of TACE or TNFR1 may also be determined in vitro byevaluating target mRNA levels or target protein levels in, for example,human corneal epithelial cells following transfection of TACE- orTNFR1-interfering RNA as described infra.

Inhibition of TNFα activity due to inhibition of TACE mRNA expression orof TNFR1 mRNA expression is also inferred in a human or mammal byobserving an improvement in a TNFα-related condition symptom such asimprovement in symptoms related to dry eye, allergic conjunctivitis,ocular inflammation, dermatitis, rhinitis, or asthma. Improvement in anyof dry eye symptoms, edema, itching, inflammation, or tolerance toenvironmental challenges, for example, is indicative of inhibition ofTNFα activity.

Interfering RNA:

In one embodiment of the invention, interfering RNA (e.g., siRNA) has asense strand and an antisense strand, and the sense and antisensestrands comprise a region of at least near-perfect contiguouscomplementarity of at least 19 nucleotides. In a further embodiment ofthe invention, interfering RNA (e.g., siRNA) has a sense strand and anantisense strand, and the antisense strand comprises a region of atleast near-perfect contiguous complementarity of at least 19 nucleotidesto a target sequence of TACE mRNA or TNFR1 mRNA, and the sense strandcomprises a region of at least near-perfect contiguous identity of atleast 19 nucleotides with a target sequence of TACE mRNA or TNFR1 mRNA,respectively. In a further embodiment of the invention, the interferingRNA comprises a region of at least 13, 14, 15, 16, 17, or 18 contiguousnucleotides having percentages of sequence complementarity to or, havingpercentages of sequence identity with, the penultimate 13, 14, 15, 16,17, or 18 nucleotides, respectively, of the 3′ end of the correspondingtarget sequence within an mRNA.

The length of each strand of the interfering RNA comprises 19 to 49nucleotides, and may comprise a length of 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, or 49 nucleotides.

The antisense strand of an siRNA is the active guiding agent of thesiRNA in that the antisense strand is incorporated into RISC, thusallowing RISC to identify target mRNAs with at least partialcomplementarity to the antisense siRNA strand for cleavage ortranslational repression.

In embodiments of the present invention, interfering RNA targetsequences (e.g., siRNA target sequences) within a target mRNA sequenceare selected using available design tools. Interfering RNAscorresponding to a TACE or TNFR1 target sequence are then tested bytransfection of cells expressing the target mRNA followed by assessmentof knockdown as described above.

Techniques for selecting target sequences for siRNAs are provided byTuschl, T. et al., “The siRNA User Guide,” revised May 6, 2004,available on the Rockefeller University web site; by Technical Bulletin#506, “siRNA Design Guidelines,” Ambion Inc. at Ambion's web site; andby other web-based design tools at, for example, the Invitrogen,Dharmacon, Integrated DNA Technologies, Genscript, or Proligo web sites.Initial search parameters can include G/C contents between 35% and 55%and siRNA lengths between 19 and 27 nucleotides. The target sequence maybe located in the coding region or in the 5′ or 3′ untranslated regionsof the mRNAs.

An embodiment of a 19-nucleotide DNA target sequence for TACE mRNA ispresent at nucleotides 297 to 315 of SEQ ID NO:1:

SEQ ID NO: 3 5′-GCTCTCAGACTACGATATT-3′.An siRNA of the invention for targeting a corresponding mRNA sequence ofSEQ ID NO:3 and having 21-nucleotide strands and a 2-nucleotide 3′overhang is:

SEQ ID NO: 4 5′-GCUCUCAGACUACGAUAUUNN-3′ SEQ ID NO: 53′-NNCGAGAGUCUGAUGCUAUAA-5′.Each “N” residue can be any nucleotide (A, C, G, U, T) or modifiednucleotide. The 3′ end can have a number of “N” residues between andincluding 1, 2, 3, 4, 5, and 6. The “N” residues on either strand can bethe same residue (e.g., UU, AA, CC, GG, or TT) or they can be different(e.g., AC, AG, AU, CA, CG, CU, GA, GC, GU, UA, UC, or UG). The 3′overhangs can be the same or they can be different. In one embodiment,both strands have a 3′UU overhang.

An siRNA of the invention for targeting a corresponding mRNA sequence ofSEQ ID NO:3 and having 21-nucleotide strands and a 3′UU overhang on eachstrand is:

SEQ ID NO: 6 5′-GCUCUCAGACUACGAUAUUUU-3′ SEQ ID NO: 73′-UUCGAGAGUCUGAUGCUAUAA-5′.

The interfering RNA may also have a 5′ overhang of nucleotides or it mayhave blunt ends. An siRNA of the invention for targeting a correspondingmRNA sequence of SEQ ID NO:3 and having 19-nucleotide strands and bluntends is:

SEQ ID NO: 8 5′-GCUCUCAGACUACGAUAUU-3′ SEQ ID NO: 93′-CGAGAGUCUGAUGCUAUAA-5′.

The strands of a double-stranded interfering RNA (e.g., an siRNA) may beconnected to form a hairpin or stem-loop structure (e.g., an shRNA). AnshRNA of the invention targeting a corresponding mRNA sequence of SEQ IDNO:3 and having a 19 bp double-stranded stem region and a 3′UU overhangis:

N is a nucleotide A, T, C, G, U, or a modified form known by one ofordinary skill in the art. The number of nucleotides N in the loop is anumber between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9,or 9 to 11, or the number of nucleotides N is 9. Some of the nucleotidesin the loop can be involved in base-pair interactions with othernucleotides in the loop. Examples of oligonucleotide sequences that canbe used to form the loop include 5′-UUCAAGAGA-3′ (Brummelkamp, T. R. etal. (2002) Science 296: 550) and 5′-UUUGUGUAG-3′ (Castanotto, D. et al.(2002) RNA 8:1454). It will be recognized by one of skill in the artthat the resulting single chain oligonucleotide forms a stem-loop orhairpin structure comprising a double-stranded region capable ofinteracting with the RNAi machinery.

The siRNA target sequence identified above can be extended at the 3′ endto facilitate the design of dicer-substrate 27-mer duplexes. Extensionof the 19-nucleotide DNA target sequence (SEQ ID NO:3) identified in theTACE DNA sequence (SEQ ID NO:1) by 6 nucleotides yields a 25-nucleotideDNA target sequence present at nucleotides 297 to 321 of SEQ ID NO:1:

SEQ ID NO: 11 5′-GCTCTCAGACTACGATATTCTCTCT-3′.A dicer-substrate 27-mer duplex of the invention for targeting acorresponding mRNA sequence of SEQ ID NO:11 is:

SEQ ID NO: 12 5′-GCUCUCAGACUACGAUAUUCUCUCU-3′ SEQ ID NO: 133′-UUCGAGAGUCUGAUGCUAUAAGAGAGA-5′.The two nucleotides at the 3′ end of the sense strand (i.e., the CUnucleotides of SEQ ID NO:12) may be deoxynucleotides for enhancedprocessing. Design of dicer-substrate 27-mer duplexes from 19-21nucleotide target sequences, such as provided herein, is furtherdiscussed by the Integrated DNA Technologies (IDT) website and by Kim,D.-H. et al., (February, 2005) Nature Biotechnology 23:2; 222-226.

When interfering RNAs are produced by chemical synthesis,phosphorylation at the 5′ position of the nucleotide at the 5′ end ofone or both strands (when present) can enhance siRNA efficacy andspecificity of the bound RISC complex but is not required sincephosphorylation can occur intracellularly.

Table 1 lists examples of TACE DNA target sequences of SEQ ID NO:1 fromwhich siRNAs of the present invention are designed in a manner as setforth above. TACE encodes tumor necrosis factor α converting enzyme, asnoted above.

TABLE 1 TACE Target Sequences for siRNAs # of Starting Nucleotide withreference to TACE Target Sequence SEQ ID NO: 1 SEQ ID NO:GCTCTCAGACTACGATATT 297 3 CCAGCAGCATTCGGTAAGA 333 14 CAGCAGCATTCGGTAAGAA334 15 AGCAGCATTCGGTAAGAAA 335 16 AGAGATCTACAGACTTCAA 355 17GAAAGCGAGTACACTGTAA 493 18 CCATGAAGAACACGTGTAA 842 19GAAGAACACGTGTAAATTA 846 20 ATCATCGCTTCTACAGATA 878 21AGAGCAATTTAGCTTTGAT 1137 22 GGTTTGACGAGCACAAAGA 1330 23TGATCCGGATGGTCTAGCA 1428 24 GCGATCACGAGAACAATAA 1508 25GCAGTAAACAATCAATCTA 1541 26 CAATCTATAAGACCATTGA 1553 27TTTCAAGAACGCAGCAATA 1591 28 TTCAAGAACGCAGCAATAA 1592 29TCAAGAACGCAGCAATAAA 1593 30 TCATGTATCTGAACAACGA 1661 31ACAGCGACTGCACGTTGAA 1691 32 GATTAATGCTACTTGCAAA 1794 33CTGGAGTCCTGTGCATGTA 1945 34 TGGAGTCCTGTGCATGTAA 1946 35GGAGTCCTGTGCATGTAAT 1947 36 CATGTAATGAAACTGACAA 1958 37CTATGTCGATGCTGAACAA 2022 38 CAAATGTGAGAAACGAGTA 2100 39GCATCGGTTCGCATTATCA 2347 40 ATCGGTTCGCATTATCAAA 2349 41CCAAGTCATTTGAGGATCT 2549 42 CCGGTCACCAGAAGTGAAA 2578 43AAAGGCTGCCTCCTTTAAA 2595 44 TTTAAACTGCAGCGTCAGA 2608 45AGATGCTGGTCATGTGTTT 2764 46 ATGCTGGTCATGTGTTTGA 2766 47TGCTGGTCATGTGTTTGAA 2767 48 CTGGTCATGTGTTTGAACT 2769 49TGTAATGAACCGCTGAATA 3027 50 GTAATGAACCGCTGAATAT 3028 51CTAAGACTAATGCTCTCTA 3261 52 AGACTAATGCTCTCTAGAA 3264 53CCTAACCACCTACCTTACA 3284 54 TACATGGTAGCCAGTTGAA 3313 55TGGTAGCCAGTTGAATTTA 3317 56 TTTATGGAATCTACCAACT 3332 57GGAATCTACCAACTGTTTA 3337 58 CATCAAGTACTGAACGTTT 434 155TCGTGGTGGTGGATGGTAA 470 156 GAAAGCGAGTACACTGTAA 493 157GAGCCTGACTCTAGGGTTC 547 158 CCACATAAGAGATGATGAT 570 159CATAAGAGATGATGATGTT 573 160 CGAATATAACATAGAGCCA 618 161GTTAATGATACCAAAGACA 649 162 CTGAAGATATCAAGAATGT 689 163ATGAAGAGTTGCTCCCAAA 755 164 ATGAAGAACACGTGTAAAT 844 165AATTATTGGTGGTAGCAGA 860 166 ATCATCGCTTCTACAGATA 878 167ATACATGGGCAGAGGGGAA 894 168 GGGCAGAGGGGAAGAGAGT 900 169GGAAGAGAGTACAACTACA 909 170 GAAGAGAGTACAACTACAA 910 171GAGAGTACAACTACAAATT 913 172 GCTAATTGACAGAGTTGAT 942 173CGGAACACTTCATGGGATA 970 174 GGATAATGCAGGTTTTAAA 984 175AGGCTATGGAATACAGATA 1002 176 GAATACAGATAGAGCAGAT 1010 177GGTAAAACCTGGTGAAAAG 1053 178 GTGAAAAGCACTACAACAT 1064 179GAGGAAGCATCTAAAGTTT 1162 180 TATGGGAACTCTTGGATTA 1215 181TGACGAGCACAAAGAATTA 1334 182 GCACAAAGAATTATGGTAA 1340 183GGTTACAACTCATGAATTG 1386 184 ACTCATGAATTGGGACATA 1393 185GTGGCGATCACGAGAACAA 1505 186 CTATAAGACCATTGAAAGT 1557 187GAACGCAGCAATAAAGTTT 1597 188 GCAATAAAGTTTGTGGGAA 1604 189CAATAAAGTTTGTGGGAAC 1605 190 GAGGGTGGATGAAGGAGAA 1626 191GGATGAAGGAGAAGAGTGT 1632 192 GCATCATGTATCTGAACAA 1658 193CAGGAAATGCTGAAGATGA 1856 194 GAATGGCAAATGTGAGAAA 2094 195GGATGTAATTGAACGATTT 2121 196 GTGGATAAGAAATTGGATA 2263 197GGATAAACAGTATGAATCT 2277 198 CCTTTAAACTGCAGCGTCA 2606 199CGTGTTGACAGCAAAGAAA 2629 200 GCAAAGAAACAGAGTGCTA 2639 201Table 2 lists examples of TNFR1 DNA target sequences of SEQ ID NO:2 fromwhich siRNAs of the present invention are designed in a manner as setforth above. TNFR1 encodes tumor necrosis factor α receptor-1, as notedabove.

TABLE 2 TNFR1 Target Sequences for siRNAs # of Starting Nucleotide withreference to TNFR1 Target Sequence SEQ ID NO: 2 SEQ ID NO:ACCAGGCCGTGATCTCTAT 124 59 AATTCGATTTGCTGTACCA 444 60TCGATTTGCTGTACCAAGT 447 61 ACAAAGGAACCTACTTGTA 469 62GAACCTACTTGTACAATGA 475 63 CTACTTGTACAATGACTGT 479 64TGTGAGAGCGGCTCCTTCA 531 65 TCAGGTGGAGATCTCTTCT 611 66CAGGTGGAGATCTCTTCTT 612 67 AGAACCAGTACCGGCATTA 667 68GAACCAGTACCGGCATTAT 668 69 AACCAGTACCGGCATTATT 669 70CCGGCATTATTGGAGTGAA 677 71 CGGCATTATTGGAGTGAAA 678 72AGCCTGGAGTGCACGAAGT 843 73 CTCCTCTTCATTGGTTTAA 960 74TTGGTTTAATGTATCGCTA 970 75 GTTTAATGTATCGCTACCA 973 76TTTAATGTATCGCTACCAA 974 77 AGTCCAAGCTCTACTCCAT 1000 78GAGCTTGAAGGAACTACTA 1053 79 CTTGAAGGAACTACTACTA 1056 80TTGAAGGAACTACTACTAA 1057 81 ACAAGCCACAGAGCCTAGA 1318 82TGTACGCCGTGGTGGAGAA 1357 83 CCGTTGCGCTGGAAGGAAT 1383 84TCTAAGGACCGTCCTGCGA 1671 85 CTAATAGAAACTTGGCACT 2044 86TAATAGAAACTTGGCACTC 2045 87 AATAGAAACTTGGCACTCC 2046 88ATAGAAACTTGGCACTCCT 2047 89 TAGAAACTTGGCACTCCTG 2048 90ATAGCAAGCTGAACTGTCC 2089 91 TAGCAAGCTGAACTGTCCT 2090 92AGCAAGCTGAACTGTCCTA 2091 93 GCAAGCTGAACTGTCCTAA 2092 94TGAACTGTCCTAAGGCAGG 2098 95 CAAAGGAACCTACTTGTAC 470 96GAGCTTGAAGGAACTACTA 1053 97 CACAGAGCCTAGACACTGA 1324 98TCCAAGCTCTACTCCATTG 1002 99 TGGAGCTGTTGGTGGGAAT 328 100GACAGGGAGAAGAGAGATA 387 101 GGGAGAAGAGAGATAGTGT 391 102GAGAAGAGAGATAGTGTGT 393 103 GAAGAGAGATAGTGTGTGT 395 104GTGTGTGTCCCCAAGGAAA 406 105 GAAAATATATCCACCCTCA 421 106AAATATATCCACCCTCAAA 423 107 CTGTACCAAGTGCCACAAA 455 108ACCAAGTGCCACAAAGGAA 459 109 CCAAGTGCCACAAAGGAAC 460 110CCACAAAGGAACCTACTTG 467 111 CAAAGGAACCTACTTGTAC 470 112AAAGGAACCTACTTGTACA 471 113 GATACGGACTGCAGGGAGT 513 114CGGACTGCAGGGAGTGTGA 517 115 TCCTTCACCGCTTCAGAAA 543 116CAGAAAACCACCTCAGACA 556 117 TGCCTCAGCTGCTCCAAAT 576 118CTCCAAATGCCGAAAGGAA 587 119 TCCAAATGCCGAAAGGAAA 588 120CCAAATGCCGAAAGGAAAT 589 121 GCCGAAAGGAAATGGGTCA 595 122AGGAAATGGGTCAGGTGGA 601 123 GGAAATGGGTCAGGTGGAG 602 124GTGTGTGGCTGCAGGAAGA 651 125 GGAAGAACCAGTACCGGCA 664 126CCATGCAGGTTTCTTTCTA 785 127 CATGCAGGTTTCTTTCTAA 786 128TGCAGGTTTCTTTCTAAGA 788 129 AGGTTTCTTTCTAAGAGAA 791 130GGTTTCTTTCTAAGAGAAA 792 131 AGAGAAAACGAGTGTGTCT 804 132GAGTGTGTCTCCTGTAGTA 813 133 CTGTAGTAACTGTAAGAAA 824 134AGAAAAGCCTGGAGTGCAC 838 135 TTGAGAATGTTAAGGGCAC 877 136TGTTAAGGGCACTGAGGAC 884 137 GGTCATTTTCTTTGGTCTT 929 138CCTCCTCTTCATTGGTTTA 959 139 TCCTCTTCATTGGTTTAAT 961 140CTCTTCATTGGTTTAATGT 963 141 TCTTCATTGGTTTAATGTA 964 142CTTCATTGGTTTAATGTAT 965 143 TCCAAGCTCTACTCCATTG 1002 144CTCCATTGTTTGTGGGAAA 1013 145 GGGAAATCGACACCTGAAA 1026 146TGAAGGAACTACTACTAAG 1058 147 ACCTCCAGCTCCACCTATA 1161 148CCCACAAGCCACAGAGCCT 1315 149 ACGCCGTGGTGGAGAACGT 1360 150GGAAGGAATTCGTGCGGCG 1393 151 TGAGCGACCACGAGATCGA 1420 152GCGAGGCGCAATACAGCAT 1471 153 TGGGCTGCCTGGAGGACAT 1573 154

As cited in the examples above, one of skill in the art is able to usethe target sequence information provided in Tables 1 or 2 to designinterfering RNAs having a length shorter or longer than the sequencesprovided in the tables and by referring to the sequence position in SEQID NO:1 or SEQ ID NO:2 and adding or deleting nucleotides complementaryor near complementary to SEQ ID NO:1 or SEQ ID NO:2, respectively.

The target RNA cleavage reaction guided by siRNAs and other forms ofinterfering RNA is highly sequence specific. In general, siRNAcontaining a sense nucleotide strand identical in sequence to a portionof the target mRNA and an antisense nucleotide strand exactlycomplementary to a portion of the target mRNA are siRNA embodiments forinhibition of mRNAs cited herein. However, 100% sequence complementaritybetween the antisense siRNA strand and the target mRNA, or between theantisense siRNA strand and the sense siRNA strand, is not required topractice the present invention. Thus, for example, the invention allowsfor sequence variations that might be expected due to genetic mutation,strain polymorphism, or evolutionary divergence.

In one embodiment of the invention, the antisense strand of the siRNAhas at least near-perfect contiguous complementarity of at least 19nucleotides with the target mRNA.

“Near-perfect,” as used herein, means the antisense strand of the siRNAis “substantially complementary to,” and the sense strand of the siRNAis “substantially identical” to at least a portion of the target mRNA.“Identity,” as known by one of ordinary skill in the art, is the degreeof sequence relatedness between nucleotide sequences as determined bymatching the order and identity of nucleotides between the sequences. Inone embodiment, the antisense strand of an siRNA having 80% and between80% up to 100% complementarity, for example, 85%, 90% or 95%complementarity, to the target mRNA sequence are considered near-perfectcomplementarity and may be used in the present invention. “Perfect”contiguous complementarity is standard Watson-Crick base pairing ofadjacent base pairs. “At least near-perfect” contiguous complementarityincludes “perfect” complementarity as used herein. Computer methods fordetermining identity or complementarity are designed to identify thegreatest degree of matching of nucleotide sequences, for example, BLASTN(Altschul, S. F., et al. (1990) J. Mol. Biol. 215:403-410).

The relationship between a target mRNA (sense strand) and one strand ofan siRNA (the sense strand) is that of identity. The sense strand of ansiRNA is also called a passenger strand, if present. The relationshipbetween a target mRNA (sense strand) and the other strand of an siRNA(the antisense strand) is that of complementarity. The antisense strandof an siRNA is also called a guide strand.

The penultimate base in a nucleic acid sequence that is written in a 5′to 3′ direction is the next to the last base, i.e., the base next to the3′ base. The penultimate 13 bases of a nucleic acid sequence written ina 5′ to 3′ direction are the last 13 bases of a sequence next to the 3′base and not including the 3′ base. Similarly, the penultimate 14, 15,16, 17, or 18 bases of a nucleic acid sequence written in a 5′ to 3′direction are the last 14, 15, 16, 17, or 18 bases of a sequence,respectively, next to the 3′ base and not including the 3′ base.

In an embodiment of the invention, the region of contiguous nucleotidesis a region of at least 13 contiguous nucleotides having at least 90%sequence complementarity to, or at least 90% sequence identity with, thepenultimate 13 nucleotides of the 3′ end of an mRNA corresponding to thesequence identified by each sequence identifier.

In one embodiment of the invention, the region of contiguous nucleotidesis a region of at least 14 contiguous nucleotides having at least 85%sequence complementarity to, or at least 85% sequence identity with, thepenultimate 14 nucleotides of the 3′ end of an mRNA corresponding to thesequence identified by each sequence identifier. Two nucleotidesubstitutions (i.e., 12/14=86% identity/complementarity) are included insuch a phrase.

In a further embodiment of the invention, the region of contiguousnucleotides is a region of at least 15, 16, 17, or 18 contiguousnucleotides having at least 80% sequence complementarity to, or at least80% sequence identity with, the penultimate 14 nucleotides of the 3′ endof an mRNA corresponding to the sequence of the sequence identifier.Three nucleotide substitutions are included in such a phrase.

The target sequence in the mRNAs corresponding to SEQ ID NO:1 or SEQ IDNO:2 may be in the 5′ or 3′ untranslated regions of the mRNA as well asin the coding region of the mRNA.

One or both of the strands of double-stranded interfering RNA may have a3′ overhang of from 1 to 6 nucleotides, which may be ribonucleotides ordeoxyribonucleotides or a mixture thereof. The nucleotides of theoverhang are not base-paired. In one embodiment of the invention, theinterfering RNA comprises a 3′ overhang of TT or UU. In anotherembodiment of the invention, the interfering RNA comprises at least oneblunt end. The termini usually have a 5′ phosphate group or a 3′hydroxyl group. In other embodiments, the antisense strand has a 5′phosphate group, and the sense strand has a 5′ hydroxyl group. In stillother embodiments, the termini are further modified by covalent additionof other molecules or functional groups.

The sense and antisense strands of the double-stranded siRNA may be in aduplex formation of two single strands as described above or may be asingle molecule where the regions of complementarity are base-paired andare covalently linked by a hairpin loop so as to form a single strand.It is believed that the hairpin is cleaved intracellularly by a proteintermed dicer to form an interfering RNA of two individual base-pairedRNA molecules.

Interfering RNAs may differ from naturally-occurring RNA by theaddition, deletion, substitution or modification of one or morenucleotides. Non-nucleotide material may be bound to the interferingRNA, either at the 5′ end, the 3′ end, or internally. Such modificationsare commonly designed to increase the nuclease resistance of theinterfering RNAs, to improve cellular uptake, to enhance cellulartargeting, to assist in tracing the interfering RNA, to further improvestability, or to reduce the potential for activation of the interferonpathway. For example, interfering RNAs may comprise a purine nucleotideat the ends of overhangs. Conjugation of cholesterol to the 3′ end ofthe sense strand of an siRNA molecule by means of a pyrrolidine linker,for example, also provides stability to an siRNA.

Further modifications include a 3′ terminal biotin molecule, a peptideknown to have cell-penetrating properties, a nanoparticle, apeptidomimetic, a fluorescent dye, or a dendrimer, for example.

Nucleotides may be modified on their base portion, on their sugarportion, or on the phosphate portion of the molecule and function inembodiments of the present invention. Modifications includesubstitutions with alkyl, alkoxy, amino, deaza, halo, hydroxyl, thiolgroups, or a combination thereof, for example. Nucleotides may besubstituted with analogs with greater stability such as replacing aribonucleotide with a deoxyribonucleotide, or having sugar modificationssuch as 2′ OH groups replaced by 2′ amino groups, 2′ O-methyl groups, 2′methoxyethyl groups, or a 2′-O, 4′-C methylene bridge, for example.Examples of a purine or pyrimidine analog of nucleotides include axanthine, a hypoxanthine, an azapurine, a methylthioadenine,7-deaza-adenosine and O- and N-modified nucleotides. The phosphate groupof the nucleotide may be modified by substituting one or more of theoxygens of the phosphate group with nitrogen or with sulfur(phosphorothioates). Modifications are useful, for example, to enhancefunction, to improve stability or permeability, or to directlocalization or targeting.

There may be a region or regions of the antisense interfering RNA strandthat is (are) not complementary to a portion of SEQ ID NO:1 or SEQ IDNO:2. Non-complementary regions may be at the 3′, 5′ or both ends of acomplementary region or between two complementary regions.

Interfering RNAs may be generated exogenously by chemical synthesis, byin vitro transcription, or by cleavage of longer double-stranded RNAwith dicer or another appropriate nuclease with similar activity.Chemically synthesized interfering RNAs, produced from protectedribonucleoside phosphoramidites using a conventional DNA/RNAsynthesizer, may be obtained from commercial suppliers such as AmbionInc. (Austin, Tex.), Invitrogen (Carlsbad, Calif.), or Dharmacon(Lafayette, Colo.). Interfering RNAs are purified by extraction with asolvent or resin, precipitation, electrophoresis, chromatography, or acombination thereof, for example. Alternatively, interfering RNA may beused with little if any purification to avoid losses due to sampleprocessing.

Interfering RNAs can also be expressed endogenously from plasmid orviral expression vectors or from minimal expression cassettes, forexample, PCR generated fragments comprising one or more promoters and anappropriate template or templates for the interfering RNA. Examples ofcommercially available plasmid-based expression vectors for shRNAinclude members of the pSilencer series (Ambion, Austin, Tex.) andpCpG-siRNA (InvivoGen, San Diego, Calif.). Viral vectors for expressionof interfering RNA may be derived from a variety of viruses includingadenovirus, adeno-associated virus, lentivirus (e.g., HIV, FIV, andEIAV), and herpes virus. Examples of commercially available viralvectors for shRNA expression include pSilencer adeno (Ambion, Austin,Tex.) and pLenti6/BLOCK-iT™-DEST (Invitrogen, Carlsbad, Calif.).Selection of viral vectors, methods for expressing the interfering RNAfrom the vector and methods of delivering the viral vector are withinthe ordinary skill of one in the art. Examples of kits for production ofPCR-generated shRNA expression cassettes include Silencer Express(Ambion, Austin, Tex.) and siXpress (Mirus, Madison, Wis.). A firstinterfering RNA may be administered via in vivo expression from a firstexpression vector capable of expressing the first interfering RNA and asecond interfering RNA may be administered via in vivo expression from asecond expression vector capable of expressing the second interferingRNA, or both interfering RNAs may be administered via in vivo expressionfrom a single expression vector capable of expressing both interferingRNAs.

Interfering RNAs may be expressed from a variety of eukaryotic promotersknown to those of ordinary skill in the art, including pol IIIpromoters, such as the U6 or H1 promoters, or pol II promoters, such asthe cytomegalovirus promoter. Those of skill in the art will recognizethat these promoters can also be adapted to allow inducible expressionof the interfering RNA.

Hybridization Under Physiological Conditions:

In certain embodiments of the present invention, an antisense strand ofan interfering RNA hybridizes with an mRNA in vivo as part of the RISCcomplex.

For example, high stringency conditions could occur at about 50%formamide at 37° C. to 42° C. Reduced stringency conditions could occurat about 35% to 25% formamide at 30° C. to 35° C. Examples of stringencyconditions for hybridization are provided in Sambrook, J., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. Further examples of stringenthybridization conditions include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing, orhybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamidefollowed by washing at 70° C. in 0.3×SSC, or hybridization at 70° C. in4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at 67° C. in1×SSC. The temperature for hybridization is about 5-10° C. less than themelting temperature (T_(m)) of the hybrid where T_(m) is determined forhybrids between 19 and 49 base pairs in length using the followingcalculation: T_(m)° C.=81.5+16.6(log₁₀[Na+])+0.41 (% G+C)−(600/N) whereN is the number of bases in the hybrid, and [Na+] is the concentrationof sodium ions in the hybridization buffer.

The above-described in vitro hybridization assay provides a method ofpredicting whether binding between a candidate siRNA and a target willhave specificity. However, in the context of the RISC complex, specificcleavage of a target can also occur with an antisense strand that doesnot demonstrate high stringency for hybridization in vitro.

Single-Stranded Interfering RNA:

As cited above, interfering RNAs ultimately function as single strands.Single-stranded (ss) interfering RNA has been found to effect mRNAsilencing, albeit less efficiently than double-stranded RNA. Therefore,embodiments of the present invention also provide for administration ofa ss interfering RNA that hybridizes under physiological conditions to aportion of SEQ ID NO:1 or SEQ ID NO:2 and has a region of at leastnear-perfect contiguous complementarity of at least 19 nucleotides withthe hybridizing portion of SEQ ID NO:1 or SEQ ID NO:2, respectively. Thess interfering RNA of Table 1 or Table 2 has a length of 19 to 49nucleotides as for the ds interfering RNA cited above. The ssinterfering RNA has a 5′ phosphate or is phosphorylated in situ or invivo at the 5′ position. The term “5′ phosphorylated” is used todescribe, for example, polynucleotides or oligonucleotides having aphosphate group attached via ester linkage to the C5 hydroxyl of thesugar (e.g., ribose, deoxyribose, or an analog of same) at the 5′ end ofthe polynucleotide or oligonucleotide.

SS interfering RNAs are synthesized chemically or by in vitrotranscription or expressed endogenously from vectors or expressioncassettes as for ds interfering RNAs. 5′ Phosphate groups may be addedvia a kinase, or a 5′ phosphate may be the result of nuclease cleavageof an RNA. Delivery is as for ds interfering RNAs. In one embodiment, ssinterfering RNAs having protected ends and nuclease resistantmodifications are administered for silencing. SS interfering RNAs may bedried for storage or dissolved in an aqueous solution. The solution maycontain buffers or salts to inhibit annealing or for stabilization.

Hairpin Interfering RNA:

A hairpin interfering RNA is a single molecule (e.g., a singleoligonucleotide chain) that comprises both the sense and antisensestrands of an interfering RNA in a stem-loop or hairpin structure (e.g.,a shRNA). For example, shRNAs can be expressed from DNA vectors in whichthe DNA oligonucleotides encoding a sense interfering RNA strand arelinked to the DNA oligonucleotides encoding the reverse complementaryantisense interfering RNA strand by a short spacer. If needed for thechosen expression vector, 3′ terminal T's and nucleotides formingrestriction sites may be added. The resulting RNA transcript folds backonto itself to form a stem-loop structure.

Mode of Administration:

Interfering RNA may be delivered via aerosol, buccal, dermal,intradermal, inhaling, intramuscular, intranasal, intraocular,intrapulmonary, intravenous, intraperitoneal, nasal, ocular, oral, otic,parenteral, patch, subcutaneous, sublingual, topical, or transdermaladministration, for example.

Administration may be directly to the eye by ocular tissueadministration such as periocular, conjunctival, subtenon, intracameral,intravitreal, intraocular, subretinal, subconjunctival, retrobulbar,intracanalicular, or suprachoroidal administration; by injection, bydirect application to the eye using a catheter or other placement devicesuch as a retinal pellet, intraocular insert, suppository or an implantcomprising a porous, non-porous, or gelatinous material; by topicalocular drops or ointments; or by a slow release device in the cul-de-sacor implanted adjacent to the sclera (transscleral) or within the eye.Intracameral injection may be through the cornea into the anteriorchamber to allow the agent to reach the trabecular meshwork.Intracanalicular injection may be into the venous collector channelsdraining Schlemm's canal or into Schlemm's canal.

Administration may be directly to the ear via, for example, topical oticdrops or ointments, slow release devices in the ear or implantedadjacent to the ear. Local administration includes otic intramuscular,intratympanic cavity and intracochlear injection routes ofadministration. Furthermore, agents can be administered to the inner earby placement of a gelfoam, or similar absorbent and adherent product,soaked with the interfering RNA against the window membrane of themiddle/inner ear or adjacent structure.

Administration may be directly to the lungs, via, for example, anaerosolized preparation, and by inhalation via an inhaler or anebulizer, for example.

Further modes of administration include tablets, pills, and capsules,all of which are capable of formulation by one of ordinary skill in theart.

Subject:

A subject in need of treatment for a TNFα-related condition or at riskfor developing a TNFα-related condition is a human or other mammalhaving a TNFα-related inflammatory condition or having dry eye or atrisk of developing a TNFα-related inflammatory condition or dry eye. ATNFα-related inflammatory condition includes, for example, allergicconjunctivitis, ocular inflammation, dermatitis, rhinitis, or asthmaassociated with undesired or inappropriate activity of TNFα as citedherein.

Ocular structures associated with a TNFα-related condition may includethe eye, retina, choroid, lens, cornea, trabecular meshwork, iris, opticnerve, optic nerve head, sclera, aqueous chamber, vitreous chamber,ciliary body, or posterior segment, for example.

Otic structures associated with such disorders may include the innerear, middle ear, outer ear, tympanic cavity or membrane, cochlea, orEustachian tube, for example.

Pulmonary structures associated with such disorders may include thenose, mouth, pharynx, larynx, bronchial tubes, trachea, carina (theridge separating the opening of the right and left main bronchi), andlungs, particularly the lower lungs, such as bronchioli and alveoli.

A subject may also be an otic cell, a lung cell, an ocular cell, cellculture, organ or an ex vivo organ or tissue.

Formulations and Dosage:

Pharmaceutical formulations comprise interfering RNAs, or salts thereof,of the invention up to 99% by weight mixed with a physiologicallyacceptable carrier medium such as water, buffer, saline, glycine,hyaluronic acid, mannitol, and the like.

Interfering RNAs of the present invention are administered as solutions,suspensions, or emulsions. The following are examples of possibleformulations embodied by this invention.

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Hydroxypropylmethylcellulose 0.5 Sodium chloride 0.8 BenzalkoniumChloride 0.01 EDTA 0.01 NaOH/HCl Qs pH 7.4 Purified water (RNase-free)Qs 100 Ml

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Phosphate Buffered Saline 1.0 Benzalkonium Chloride 0.01 Polysorbate 800.5 Purified water (RNase-free) q.s. to 100%

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Monobasic sodium phosphate 0.05 Dibasic sodium phosphate 0.15(anhydrous) Sodium chloride 0.75 Disodium EDTA 0.05 Cremophor EL 0.1Benzalkonium chloride 0.01 HCl and/or NaOH pH 7.3-7.4 Purified water(RNase-free) q.s. to 100%

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Phosphate Buffered Saline 1.0 Hydroxypropyl-β-cyclodextrin 4.0 Purifiedwater (RNase-free) q.s. to 100%

Generally, an effective amount of the interfering RNAs of embodiments ofthe invention results in an extracellular concentration at the surfaceof the target cell of from 100 pM to 100 nM, or from 1 nM to 50 nM, orfrom 5 nM to about 10 nM, or about 25 nM. The dose required to achievethis local concentration will vary depending on a number of factorsincluding the delivery method, the site of delivery, the number of celllayers between the delivery site and the target cell or tissue, whetherdelivery is local or systemic, etc. The concentration at the deliverysite may be considerably higher than it is at the surface of the targetcell or tissue. Topical compositions are delivered to the surface of thetarget organ one to four times per day, or on an extended deliveryschedule such as daily, weekly, bi-weekly, monthly, or longer, accordingto the routine discretion of a skilled clinician. The pH of theformulation is about pH 4-9, or pH 4.5 to pH 7.4.

Therapeutic treatment of patients with siRNAs directed against TACE mRNAor TNFR1 mRNA is expected to be beneficial over small moleculetreatments by increasing the duration of action, thereby allowing lessfrequent dosing and greater patient compliance.

An effective amount of a formulation may depend on factors such as theage, race, and sex of the subject, the severity of the TNFα-relatedcondition, the rate of target gene transcript/protein turnover, theinterfering RNA potency, and the interfering RNA stability, for example.In one embodiment, the interfering RNA is delivered topically to atarget organ and reaches TACE mRNA- or TNFR1 mRNA-containing tissue at atherapeutic dose thereby ameliorating a TNFα-related process.

Acceptable Carriers:

An acceptable carrier refers to those carriers that cause at most,little to no ocular irritation, provide suitable preservation if needed,and deliver one or more interfering RNAs of the present invention in ahomogenous dosage. An acceptable carrier for administration ofinterfering RNA of embodiments of the present invention include thecationic lipid-based transfection reagents TransIT®-TKO (MinisCorporation, Madison, Wis.), LIPOFECTIN®, Lipofectamine, OLIGOFECTAMINE™(Invitrogen, Carlsbad, Calif.), or DHARMAFECT™ (Dharmacon, Lafayette,Colo.); polycations such as polyethyleneimine; cationic peptides such asTat, polyarginine, or Penetratin (Antp peptide); or liposomes. Liposomesare formed from standard vesicle-forming lipids and a sterol, such ascholesterol, and may include a targeting molecule such as a monoclonalantibody having binding affinity for endothelial cell surface antigens,for example. Further, the liposomes may be PEGylated liposomes.

The interfering RNAs may be delivered in solution, in suspension, or inbioerodible or non-bioerodible delivery devices. The interfering RNAscan be delivered alone or as components of defined, covalent conjugates.The interfering RNAs can also be complexed with cationic lipids,cationic peptides, or cationic polymers; complexed with proteins, fusionproteins, or protein domains with nucleic acid binding properties (e.g.,protamine); or encapsulated in nanoparticles. Tissue- or cell-specificdelivery can be accomplished by the inclusion of an appropriatetargeting moiety such as an antibody or antibody fragment.

For ophthalmic, otic, or pulmonary delivery, an interfering RNA may becombined with ophthalmologically, optically, or pulmonary acceptablepreservatives, co-solvents, surfactants, viscosity enhancers,penetration enhancers, buffers, sodium chloride, or water to form anaqueous, sterile suspension or solution. Solution formulations may beprepared by dissolving the interfering RNA in a physiologicallyacceptable isotonic aqueous buffer. Further, the solutions may includean acceptable surfactant to assist in dissolving the inhibitor.Viscosity building agents, such as hydroxymethyl cellulose, hydroxyethylcellulose, methylcellulose, polyvinylpyrrolidone, or the like may beadded to the compositions of the present invention to improve theretention of the compound.

In order to prepare a sterile ointment formulation, the interfering RNAis combined with a preservative in an appropriate vehicle, such asmineral oil, liquid lanolin, or white petrolatum. Sterile gelformulations may be prepared by suspending the interfering RNA in ahydrophilic base prepared from the combination of, for example,CARBOPOL°-940 (BF Goodrich, Charlotte, N.C.), or the like, according tomethods known in the art. VISCOAT® (Alcon Laboratories, Inc., FortWorth, Tex.) may be used for intraocular injection, for example. Othercompositions of the present invention may contain penetration enhancingagents such as cremephor and TWEEN® 80 (polyoxyethylene sorbitanmonolaureate, Sigma Aldrich, St. Louis, Mo.), in the event theinterfering RNA is less penetrating in the organ or tissue of interest.

Kits:

Embodiments of the present invention provide a kit that includesreagents for attenuating the expression of an mRNA as cited herein in acell. The kit contains an siRNA or an shRNA expression vector. ForsiRNAs and non-viral shRNA expression vectors the kit also may contain atransfection reagent or other suitable delivery vehicle. For viral shRNAexpression vectors, the kit may contain the viral vector and/or thenecessary components for viral vector production (e.g., a packaging cellline as well as a vector comprising the viral vector template andadditional helper vectors for packaging). The kit may also containpositive and negative control siRNAs or shRNA expression vectors (e.g.,a non-targeting control siRNA or an siRNA that targets an unrelatedmRNA). The kit also may contain reagents for assessing knockdown of theintended target gene (e.g., primers and probes for quantitative PCR todetect the target mRNA and/or antibodies against the correspondingprotein for western blots). Alternatively, the kit may comprise an siRNAsequence or an shRNA sequence and the instructions and materialsnecessary to generate the siRNA by in vitro transcription or toconstruct an shRNA expression vector.

A pharmaceutical combination in kit form is further provided thatincludes, in packaged combination, a carrier means adapted to receive acontainer means in close confinement therewith and a first containermeans including an interfering RNA composition and an acceptablecarrier. Such kits can further include, if desired, one or more ofvarious conventional pharmaceutical kit components, such as, forexample, containers with one or more pharmaceutically acceptablecarriers, additional containers, etc., as will be readily apparent tothose skilled in the art. Printed instructions, either as inserts or aslabels, indicating quantities of the components to be administered,guidelines for administration, and/or guidelines for mixing thecomponents, can also be included in the kit.

The ability of TACE- or TNFR1-interfering RNA to knock-down the levelsof endogenous TACE or TNFR1 expression in, for example, human cornealepithelial cells is evaluated in vitro as follows. Transformed humancorneal epithelial cells, for example, the CEPI-17 cell line (Offord etal. (1999) Invest Ophthalmol V is Sci. 40:1091-1101), are plated 24 hprior to transfection in KGM keratinocyte medium (Cambrex, EastRutherford, N.J.). Transfection is performed using DharmaFECT™ 1(Dharmacon, Lafayette, Colo.) according to the manufacturer'sinstructions at TACE- or TNFR1-interfering RNA concentrations rangingfrom 0.1 nM-100 nM. Non-targeting control interfering RNA and lamin A/Cinterfering RNA (Dharmacon) are used as controls. Target mRNA levels areassessed by qPCR 24 h post-transfection using, for example, TAQMAN®forward and reverse primers and a probe set that encompasses the targetsite (Applied Biosystems, Foster City, Calif.). Target protein levelsmay be assessed approximately 72 h post-transfection (actual timedependent on protein turnover rate) by western blot, for example.Standard techniques for RNA and/or protein isolation from cultured cellsare well-known to those skilled in the art. To reduce the chance ofnon-specific, off-target effects, the lowest possible concentration ofTACE- or TNFR1 interfering RNA is used that produces the desired levelof knock-down in target gene expression.

Those of skill in the art, in light of the present disclosure, willappreciate that obvious modifications of the embodiments disclosedherein can be made without departing from the spirit and scope of theinvention. All of the embodiments disclosed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. The full scope of the invention is set out in the disclosureand equivalent embodiments thereof. The specification should not beconstrued to unduly narrow the full scope of protection to which thepresent invention is entitled.

While a particular embodiment of the invention has been shown anddescribed, numerous variations and alternate embodiments will occur tothose skilled in the art. Accordingly, it is intended that the inventionbe limited only in terms of the appended claims.

The invention may be embodied in other specific forms without departingfrom its spirit or essential characteristics. The described embodimentsare to be considered in all respects only as illustrative and notrestrictive. The scope of the invention is, therefore, indicated by theappended claims rather than by the foregoing description. All changes tothe claims that come within the meaning and range of equivalency of theclaims are to be embraced within their scope. Further, all publisheddocuments, patents, and applications mentioned herein are herebyincorporated by reference, as if presented in their entirety.

Example 1

Interfering RNA for Specifically Silencing TNFR1 in GTM-3 Cells

The present study examines the ability of TNFR1 interfering RNA to knockdown the levels of endogenous TNFR1 protein expression in cultured GTM-3cells.

Transfection of GTM-3 cells (Pang, I. H. et al., 1994. Curr. Eye Res.13:51-63) was accomplished using standard in vitro concentrations(0.1-10 nM) of TNFR1 siRNAs, siCONTROL RISC-free siRNA #1, or siCONTROLNon-targeting siRNA #2 (NTC2) and DHARMAFECT® #1 transfection reagent(Dharmacon, Lafayette, Colo.). All siRNAs were dissolved in 1×siRNAbuffer, an aqueous solution of 20 mM KCl, 6 mM HEPES (pH 7.5), 0.2 mMMgCl₂. Control samples included a buffer control in which the volume ofsiRNA was replaced with an equal volume of 1×siRNA buffer (-siRNA).Western blots using an anti-TNFR1 antibody (Santa Cruz Biotechnology,Santa Cruz, Calif.) were performed to assess TNFR1 protein expression.The TNFR1 siRNAs are double-stranded interfering RNAs having specificityfor the following targets: siTNFR1 #1 targets the sequenceCAAAGGAACCUACUUGUAC (SEQ ID NO: 202); siTNFR1 #2 targets the sequenceGAGCUUGAAGGAACUACUA (SEQ ID NO: 203); siTNFR1 #3 targets the sequenceCACAGAGCCUAGACACUGA (SEQ ID NO: 204); siTNFR1 #4 targets the sequenceUCCAAGCUCUACUCCAUUG (SEQ ID NO: 205). As shown by the data in FIG. 1,siTNFR1 #1, siTNFR1 #2, and siTNFR1 #3 siRNAs reduced TNFR1 proteinexpression significantly at the 10 nM and 1 nM concentrations relativeto the control siRNAs, but exhibited reduced efficacy at 0.1 nM. ThesiTNFR1 #2 and siTNFR1 #3 siRNAs were particularly effective. ThesiTNFR1 #4 siRNA also showed a concentration dependent reduction inTNFR1 protein expression as expected.

What is claimed is:
 1. A method of attenuating expression of TNFR1 mRNAof a subject in need thereof, comprising: administering to the subject acomposition comprising an effective amount of interfering RNA having alength of 19 to 49 nucleotides and a pharmaceutically acceptablecarrier, the interfering RNA comprising: a sense nucleotide strand, anantisense nucleotide strand, and a region of at least near-perfectcontiguous complementarity of at least 19 nucleotides; wherein theantisense strand hybridizes under physiological conditions to a portionof mRNA corresponding to SEQ ID NO:2 beginning at nucleotide 124, 328,387, 391, 393, 395, 406, 421, 423, 444, 447, 455, 459, 460, 467, 469,470, 471, 475, 479, 513, 517, 531, 543, 556, 576, 587, 588, 589, 595,601, 602, 611, 612, 651, 664, 667, 668, 669, 677, 678, 785, 786, 788,791, 792, 804, 813, 824, 838, 843, 877, 884, 929, 959, 960, 961, 963,964, 965, 970, 973, 974, 1000, 1002, 1013, 1026, 1053, 1056, 1057, 1058,1161, 1315, 1318, 1357, 1360, 1383, 1393, 1420, 1471, 1573, 1671, 2044,2045, 2046, 2047, 2048, 2089, 2090, 2091, or, 2092; wherein theexpression of TNFR1 mRNA is attenuated thereby.
 2. The method of claim1, wherein the subject is a human and the human has dry eye.
 3. Themethod of claim 1, wherein the sense nucleotide strand and the antisensenucleotide strand are connected by a loop nucleotide strand.
 4. Themethod of claim 1, wherein the composition is administered via anaerosol, buccal, dermal, intradermal, inhaling, intramuscular,intranasal, intraocular, intrapulmonary, intravenous, intraperitoneal,nasal, ocular, oral, otic, parenteral, patch, subcutaneous, sublingual,topical, or transdermal route.
 5. The method of claim 1, wherein theinterfering RNA is administered via in vivo expression from anexpression vector capable of expressing the interfering RNA.
 6. A methodof treating a TNFα-related ocular condition in a subject in needthereof, comprising: administering to an eye of the subject acomposition beginning at an effective amount of interfering RNA having alength of 19 to 49 nucleotides, and a pharmaceutically acceptablecarrier, the interfering RNA comprising a sense nucleotide strand, anantisense nucleotide strand, and the sense and antisense strandscomprise a region of least near-perfect contiguous complementarity of atleast 19 nucleotides; wherein the antisense strand hybridizes underphysiological conditions to a portion of mRNA corresponding to SEQ IDNO:2 comprising nucleotide 124, 328, 387, 391, 393, 395, 406, 421, 423,444, 447, 455, 459, 460, 467, 469, 470, 471, 475, 479, 513, 517, 531,543, 556, 576, 587, 588, 589, 595, 601, 602, 611, 612, 651, 664, 667,668, 669, 677, 678, 785, 786, 788, 791, 792, 804, 813, 824, 838, 843,877, 884, 929, 959, 960, 961, 963, 964, 965, 970, 973, 974, 1000, 1002,1013, 1026, 1053, 1056, 1057, 1058, 1161, 1315, 1318, 1357, 1360, 1383,1393, 1420, 1471, 1573, 1671, 2044, 2045, 2046, 2047, 2048, 2089, 2090,2091, or, 2092; wherein the TNFα-related ocular condition is treatedthereby, and wherein the TNFα-related ocular condition is dry eye,allergic conjunctivitis or ocular inflammation.
 7. The method of claim6, further comprising administering to the eye of the subject a secondinterfering RNA having a length of 19 to 49 nucleotides and comprising:a sense nucleotide strand, an antisense nucleotide strand, and the senseand antisense strands comprise a region of least near-perfect contiguouscomplementarity of at least 19 nucleotides wherein the antisense strandof the second interfering RNA hybridizes under physiological conditionsto a portion of mRNA corresponding to SEQ ID NO:1 comprising nucleotide297, 333, 334, 335, 434, 470, 493, 547, 570, 573, 618, 649, 689, 755,842, 844, 846, 860, 878, 894, 900, 909, 910, 913, 942, 970, 984, 1002,1010, 1053, 1064, 1137, 1162, 1215, 1330, 1334, 1340, 1386, 1393, 1428,1505, 1508, 1541, 1553, 1557, 1591, 1592, 1593, 1597, 1604, 1605, 1626,1632, 1658, 1661, 1691, 1794, 1856, 1945, 1946, 1947, 1958, 2022, 2094,2100, 2121, 2263, 2277, 2347, 2349, 2549, 2578, 2595, 2606, 2608, 2629,2639, 2764, 2766, 2767, 2769, 3027, 3028, 3261, 3264, 3284, 3313, 3317,3332, or
 3337. 8. The method of claim 6, wherein the sense nucleotidestrand and the antisense nucleotide strand are connected by a loopnucleotide strand.
 9. The method of claim 6, wherein the composition isadministered via an aerosol, buccal, dermal, intradermal, inhaling,intramuscular, intranasal, intraocular, intrapulmonary, intravenous,intraperitoneal, nasal, ocular, oral, otic, parenteral, patch,subcutaneous, sublingual, topical, or transdermal route.
 10. The methodof claim 6, wherein the interfering RNA is administered via in vivoexpression from an expression vector capable of expressing theinterfering RNA.
 11. The method of claim 6, wherein each strand of thesiRNA molecule is independently about 19 nucleotides to about 25nucleotides in length.
 12. The method of claim 6, wherein each strand ofthe siRNA molecule is independently about 19 nucleotides to about 21nucleotides in length.
 13. A method of attenuating expression of TACEmRNA of a subject, comprising: administering to the subject acomposition comprising an effective amount of interfering RNA having alength of 19 to 49 nucleotides and a pharmaceutically acceptablecarrier, the interfering RNA comprising: a region of at least 13, 14,15, 16, 17, or 18 contiguous nucleotides having at least 80% sequencecomplementarity to, or at least 90% sequence identity with, thepenultimate 13 nucleotides of the 3′ end of an mRNA corresponding to anyone of SEQ ID NO:3, SEQ ID NO:14-SEQ ID NO:58, and SEQ ID NO:155-SEQ IDNO:201, wherein the expression of TACE mRNA is attenuated thereby. 14.The method of claim 13, wherein the interfering RNA is one of an shRNA,an miRNA, or an siRNA.
 15. The method of claim 13, wherein thecomposition is administered via an aerosol, buccal, dermal, intradermal,inhaling, intramuscular, intranasal, intraocular, intrapulmonary,intravenous, intraperitoneal, nasal, ocular, oral, otic, parenteral,patch, subcutaneous, sublingual, topical, or transdermal route.
 16. Themethod of claim 13, wherein the interfering RNA is administered via invivo expression from an expression vector capable of expressing theinterfering RNA.
 17. A method of treating a TNFα-related condition in asubject in need thereof, comprising: administering to the subject acomposition comprising an effective amount of interfering RNA having alength of 19 to 49 nucleotides, and a pharmaceutically acceptablecarrier, wherein the interfering RNA comprises a region of at least 13,14, 15, 16, 17, or 18 contiguous nucleotides having at least 80%sequence complementarity to, or at least 90% sequence identity with, thepenultimate 13 nucleotides of the 3′ end of an mRNA corresponding to anyone of SEQ ID NO:3, SEQ ID NO:14-SEQ ID NO:58, and SEQ ID NO:155-SEQ IDNO:201; wherein the TNFα-related condition is treated thereby, andwherein the TNFα-related ocular condition is dry eye, allergicconjunctivitis or ocular inflammation.
 18. The method of claim 17,wherein the interfering RNA is one of an shRNA, an miRNA, or an siRNA.19. The method of claim 17, wherein the composition is administered viaan aerosol, buccal, dermal, intradermal, inhaling, intramuscular,intranasal, intraocular, intrapulmonary, intravenous, intraperitoneal,nasal, ocular, oral, otic, parenteral, patch, subcutaneous, sublingual,topical, or transdermal route.
 20. The method of claim 17, wherein theinterfering RNA is administered via in vivo expression from anexpression vector capable of expressing the interfering RNA.